Supplementary information.
b
A450
A450
a
d
A450
A450
c
S1. Cytotoxicity studies in BHK (baby hamster kidney) cells. Effect of lipospermines (a) and
lipospermines/DNA (b) on the proliferation of BHK cells. The cytotoxic properties of lipidspermines were assessed by WST-8 assay (Roche), according to protocol, for in vitro
conditions with BHK cells. Concentrations of the lipid-spermine that were chosen for this
assay are the same as in the different charge ratios ranging from 0.3:1 to 3:1. Absorbance was
measure at 450  and is here represented as the absorbance of the treated cells divided by that
of untreated cells. Each point represents a mean ± S.D. of three independent experiments.
1
C
1
2
3
4
5
6
7
S2. Incomplete dissociation of spermine derivative/DNA complexes. Oleylspermine/DNA
complexes were prepared at different charge ratios and left to equilibrate at room temperature
(RT) for 30 minutes. DNA complexes were subjected to the same treatment as for DNase I
protection assay but in the absence of DNase I. Briefly, DNase I buffer (10 mM Tris-HCl, 2.5
mM MgCl2, 0.5 mM CaCl2, pH 7.02) was added and samples were incubated for 15 min at
RT. Thereafter, EDTA, pH 8.0, and SDS were added up to the final concentrations of 0.05 M
and 1% (w/v) , respectively. Samples were analyzed in agarose gel (0.8% TAE). C: untreated
plasmid DNA. Lanes 1 to 7 correspond to the different charge ratios at which the complexes
were formulated: 0.3:1, 0.6:1, 1:1, 2:1, 3:1, 5:1 and 10:1, respectively.
2
40
30
zeta potential (mv)
20
10
0
-10
-20
Decanoyl-Spermine
Oleyl-Spermine
-30
Palmitoyl-Spermine
-40
-50
Butanoyl-Spermine
3
5
7
9
11
13
15
[lipid-spermine] (1 x 10
17
19
21
23
-5
S3. Dynamic light scattering studies – zeta potential measurements. Samples were freshly
prepared in mannitol solution (5.45%) at the DNA concentration of 0.1 g/l and left at RT
to equilibrate for 20 min. Measured concentrations of lipospermines correspond to those
present in DNA complexes formed at 0.6:1, 1:1 and 2:1 charge ratios. Each point represents a
mean ± S.D. of three experiments.
3